- Email: [email protected]
The enzymic formation of acetylmethylcarbinol and related compounds
VOL. 8 (I952)
BIOCHIMICA ET BIOPHYSICA ACTA
543
T H E ENZYMIC FORMATION OF ACETYLMETHYLCARBINOL AND R E L A T E D COMPOUNDS by FRANK C. HAPPOLD AND C. P. SPENCER
Department o/Biochemistry, University o/Leeds (England)
INTRODUCTION
Acetylmethylcarbinol, 2:3 butylene glycol and diacetyl are of widespread occurrence as products of carbohydrate metabolism. The problem of the mechanism of their formation has attracted numerous workers, the first investigations being made by NEUBERG and his associates working with yeast1, 2, 3,4, 5, s, 7. They suggested that pyruvic acid was first decarboxylated (by carboxylase) to acetaldehyde which then condensed to form acetylmethylcarbinol. They postulated an enzyme "carboligase" to catalyse this reaction. Although acetylmethylcarbinol is not formed during the normal fermentation of glucose by yeast, additions of acetaldehyde (or other hydrogen acceptors) cause its formation. The anomaly that the carbinol is not formed from glucose alone was later suggested by KLUYVERAND DUNKERs to be due to the rapid removal of acetaldehyde by reduction. No satisfactory biosynthesis of acetylmethylcarbinol using acetaldehyde alone was achieved although subsequently several reports of such a reaction have appeared (ToMIYASU9,10 and KUZlNn). DIRSCHERL12,13 has suggested that the carbinol is formed by the non-enzymic condensation of active (i.e. biologically produced) acetaldehyde, a hypothesis that gained support by the failure of several workers to demonstrate a separate carboxylase and carboligase (TANKO AND MUNK14). Isotopic studies have since confirmed that acetaldehyde added to yeast fermentations is converted to acetylmethylcarbinol but they did little to elucidate the actual mechanism of its conversion (GROSSAND WERKMAN TM16). The data obtained from enzyme studies with mammalian tissue extracts are in many ways analogous to those obtained during studies with yeast. The addition of acetaldehyde to the decarboxylation of pyruvic acid by an enzyme system isolated from pigs heart causes a marked increase in the rate of both carbon dioxide and acetylmethylcarbinol production (GREEN, WESTERFIELD, VENNESLAND, AND KNOX17), and both systems are able to form acetylmethylcarbinol from acetaldehyde alone. Somewhat conflicting data have been obtained in studies with bacteria. Pyruvic acid acts as a precursor of both carbinol and glycol (BARRITTTM) and the addition of acetaldehyde (MICHELSON AND WERKMAN 19) or acetic acid (REYNOLDS AND WERKMAN 20) to fermentations of glucose by Aerobacter indologenes and Bacillus polymyxa (STAHYL AND WERKMANll) cause an increased formation of the four-carbon compounds. The position was further complicated by the preparation of a cell free enzyme system from A. aerogenes which catalyses the anaerobic decarboxylation of pyruvic acid with concurrent formation of acetylmethylcarbinol (SILVERMAN AND WERKMAN22), but does not Re#rences p. 556.
544
v . c . HAPPOLD, C. P. SPENCER
VOL. 8
(1952)
utilize acetaldehyde or acetyl phosphate during the dissimilation of pyruvate. Later isotopic studies (GRoss AND WERKMAN 15) confirmed these results and indicated that at least two mechanisms existed. It was suggested that whilst the condensation of acetaldehyde can be accomplished by whole cells this system is absent from cell-free extracts which produce aeetylmethylcarbinol by another mechanism. The studies reported in this paper are primarily concerned with the mechanism of formation of acetylmethylcarbinol by bacteria and we have endeavoured to relate the results obtained with those from studies with other systems. EXPERIMENTAL METHODS
Materials The sample of co-carboxylase used was synthesised by the method of TAUBER*S; sodium p y r u v a t e was prepared as previously described (HAPPOLD AND SPENCER~a).
Methods o] Estimation Pyruvie acid was determined colorimetrically as the 2:4 dinitrophenylhydrazone following a specific extraction procedure (FRIED~MANN AND HAUGEN~). Protein nitrogen was e s t i m a t e d b y a micro-Kjeldahl m e t h o d (MARKHAM'% Acetylmethylcarbinol was determined b y the m e t h o d described in a previous paper 14. No 2 : 3 butylene glycol was formed b y t h e system. Carbon dioxide was measured by conventional WARBURGtechnique using air as the gas phase in the flasks. Preliminary extmrim~nts showed t h a t carbon dioxide evolution was t h e only gas exchange catalysed b y the crude enzyme, oxygen uptake being zero or negligible. Carbon dioxide evolution was measured at 37 ° C, p y r u v a t e being added from the side a r m a t zero time. The m a i n chamber of the flasks n o r m a l l y contained t h e enzyme solution a n d other a d d e n d a in M[I 5 p h o s p h a t e buffer at p H 5.9. I n t h e experiments when parallel estimations of carbon dioxide and acetylmethylcarbinol were made, the carbon dioxide production was first e s t i m a t e d m a n o m e t r i c a l l y a n d immediately afterwards samples were withdrawn from t h e flasks, pipetted into a protein precipitant ( n o r m a l l y lO% trichloracetic acid) and t h e acetylmethylcarbinol determined. Parallel control experiments using a boiled sample of t h e enzyme a n d w i t h o u t addition of the s u b s t r a t e were always performed.
Growth o] Cells A. aerogenes (laboratory strain) was m a i n t a i n e d b y fortnightly sub-culturing on ordinary agar slopes. The organism was grown in bulk in io 1 quantities of the following m e d i u m : I . o % glucose, 0 . 3 % peptone, o.8% dipotassium hydrogen phosphate, i o % t a p water (metals). The t a p water and p h o s p h a t e were autoclaved separately and mixed asceptically after sterilization. The bulk m e d i u m was inoculated with t h e whole of t h e growth from a 24 hour slope culture of the organism a n d was incubated for 2o to 24 hours a t 37 ~ C. The cells were harvested in the form of a paste b y m e a n s of an Alfa Laval Centrifugal Oil Separator.
Preparation o] Cell-Free Enzyme Extracts SILVERMAN AND WERKMAN22 ground their cells with powdered glass in preparing their e x t r a c t s whilst GALE~7 shook cell suspensions with glass beads to liberate the acetylmethylcarbinol forming s y s t e m from the bacteria. Both m e t h o d s have been used in t h e present work. Active e x t r a c t s were also obtained from acetone dried bacteria prepared b y the following m e t h o d which overcomes the difficulties of drying ceils producing mucilagenous polysaccharide in their growth. The cells were grown in the usual glucose-peptone media adjusted to p H 6.0 before inoculation. The bulk m e d i u m was inoculated by transferring a loop of growth from a 24 h o u r agar slope culture of the organism to 9 ml of sterile water and agitating until the suspension became j u s t observably turbid. The bulk m e d i u m was inoculated with the whole of t h i s suspension a n d was incubated at 3 °° C for a m a x i m u m of 2o hours. These details resulted in somewhat impaired yields b u t limited the a m o u n t of polysaccharide formed by t h e cells a n d so minimised their resistance to the action of t h e acetone. Failure to observe these details always resulted in a yield of cells unsuitable for acetone drying. The cells from IO 1 of m e d i u m were harvested, washed, resuspended in 2oo ml of distilled water a n d cooled to o ° C. The chilled suspension was t h e n slowly added to 5 volumes of ice cold acetone, t h e addition being made slowly and with c o n s t a n t agitation; the suspension was stored at - 1 5 ° C for 15 to 24 hours. The cells were finally separated by filtration, washed first with 75 ° ml of 50% acetone-ether and finally with 250 ml of ether, care being taken to see t h a t the powder did not become dry until after t h e final washing with ether. Final drying was t h e n accomplished b y suction of air t h r o u g h the .filter.
Re#fences p. 556.
VOL.
8 (1952)
ENZYMIC FORMATION OF ACETYLMETHYLCARBINOL
545
A c e t o n e - d r i e d p o w d e r s of A. aerogenes p r e p a r e d in t h i s w a y were q u i t e stable if s t o r e d in a d e s i c c a t o r a t r o o m t e m p e r a t u r e . T h e e n z y m e s y s t e m w a s e x t r a c t e d w i t h M / I 5 p h o s p h a t e buffer, p H 6.0, t h e o p t i m u m t i m e of e x t r a c t i o n being 12 h o u r s a t o ° C. T h e cell debris w a s s e d i m e n t e d b y c e n t r i f u g a t i o n for 3 ° to 45 m i n u t e s a t 7ooo r.p.m. T h e a v e r a g e yield of cell free e x t r a c t f r o m io 1 of m e d i u m w a s a b o u t 15 ml.
General E n z y m e e x t r a c t s o b t a i n e d b y all t h r e e m e t h o d s q u i c k l y lost a c t i v i t y , five h o u r s s t a n d i n g a t r o o m t e m p e r a t u r e b e i n g sufficient to c a u s e i n a c t i v a t i o n . Irreversible i n a c t i v a t i o n occurred after a b o u t 3 ~ h o u r s a t 37 ° C e v e n in t h e presence of t h e s u b s t r a t e , a n d a t o ° C c o m p l e t e i n a c t i v a t i o n occurred a f t e r 3o hours. A d d i t i o n s of i n e r t proteins, p r o t e c t i v e colloids a n d r e d u c i n g a g e n t s did n o t stabilize t h e s y s t e m . T h e cell free e x t r a c t s were, therefore, stored in a frozen c o n d i t i o n a t - ! 5 ° C. SILVERMAN AND WERKMAN r e p o r t e d t h a t if frozen solid, e x t r a c t s could be s t o r e d for a considerable period w i t h l i t t l e loss of d e c a r b o x y l a t i n g a c t i v i t y . O u r experience h a s confirmed t h i s b u t h a s also s h o w n t h a t freezing a n d t h a w i n g are n o t w i t h o u t effect u p o n t h e a c e t y l m e t h y l e a r b i n o l a c t i v i t y . T h e practice of s t o r i n g e x t r a c t s before use was, therefore, avoided. EXPERIMENTAL
RESULTS
Determination o/the optimum p H o/the enzyme system A sample of the cell free extract was obtained 'by the glass grinding technique. The production of carbon dioxide and acetylmethylcarbinol from pyruvate by the enzyme preparation was measured in citrate-phosphate buffers at various pH's determined by a Glass Electrode. There was no appreciable change in the p H of any of the reactants during incubation. The results shown in Fig. I are typical of those obtained. The determinations of the optimum p H of carbon dioxide and acetylmethylcarbinol were made on different samples of the extract and the relative amounts of the two compounds formed are not comparable. The p H optimum of the system is about 5.9 for both acetylmethylcarbinol and carbon dioxide production. All extracts were inactive below a pH of 4 when protein precipitation occurred.
(o}
I
,l
(b)
30o
i
~ 2oo "~150
o
~
3.O
4.0
5.0
60
~o
~oo
3.g
4.0
~0
60
7.0
~0
Fig. I. p H a c t i v i t y c u r v e s for a c e t y l m e t h y l c a r b i n o l e n z y m e of A. aerogenes. (a) a c e t y l m e t h y l c a r b i n o i p r o d u c t i o n . (b) c a r b o n dioxide p r o d u c t i o n .
Re/erences p. 556.
546
F . C . HAPPOLD, C. P. SPENCER
VOL. 8
(1952)
Comparison o[ the cell-/ree extracts obtained by the three methods o] preparation The extracts prepared by the three methods of preparation were compared by means of an arbitrary unit of activity. This was defined as: that amount of acetylmethylcarbinol (in ~g) or carbon dioxide (in ~1) produced by an amount of enzyme containing I mg protein nitrogen after 3 ° minutes incubation at 37 ° C in M/I 5 phosphate buffer at pH 5.9 in the presence of excess pyruvate. The results place the shaking method as the most efficient but the extracts obtained from the acetone dried powders are the most convenient to prepare, and such extracts have been used in all further studies unless it is stated otherwise. In the course of these studies, whilst comparing KC1 with the M/I 5 phosphate as an extraction agent for the enzyme system, it was observed that variations in the strength of the KC1 employed (from 1/s to ~ saturation) caused COs evolution to drop from IiO to 32 /A/3 o min/mg N~, whereas acetylmethylcarbinol production fell only from 290 to 260/~g.
The effect o] co-carboxylase, manganese and magnesium ions on the enzyme system SILVERMAN AND WERKMAN reported that co-carboxylase, manganese and, to a lesser extent, magnesium ions, increase the rate of decarboxylation of pyruvate. These effects were reinvestigated by measuring the effects of these ions on both carbon dioxide and acetylmethylcarbinol production. Saturation of the system with either co-carboxylase or manganese ions stimulates the initial rate of carbon dioxide and of acetylmethylcarbinol production. The observation that magnesium ions acted similarly could not be confirmed. Co-carboxylase alone causes a disproportionate stimulation of the initial rate of carbon dioxide production. In fresh samples of the extract equality in the magnitude of the effect on the two activities is restored by concurrent saturation of the system with manganese ions. It proved impossible to resolve the system by dialysis (with a view to its subsequent reconstruction from known dialysable factors) since, owing to its instability at temperatures above its freezing point, irreversible inactivation of the protein moiety occurs. Dialysable factors are, however, involved since a dialysate (obtained by dialysis of a cell free extract against distilled water) after concentration in a vacuum at 25 ° C shows a stimulating action on the initial rates of both carbon dioxide and acetylmethylcarbinol production.
Adsorption o/the enzyme complex When an active phosphate buffer extract of acetone dried powder is shaken with kieselguhr, the supernatant removed and the kieselguhr washed with phosphate buffer at pH 5.9 an interesting phenomenon occurs. The carbon dioxide activity of the control is 430 ~1/3o min/o.5 ml, the equivalent of the supernatant and the washings 322 and I21 ~1 respectively. Apparently no adsorption takes place and the agreement between the control and the summated fractions is excellent. When aliquots of the same preparations are tested for acetylmethylcarbinol, however, the summated activity of the fractions are apparently greater than the starting material alone, moreover, the acetylmethylcarbinol/carbon dioxide ratio (~M/ml) changes from 1.29 with the control material to 1.63 with the supernatant and 2.1 with the washings. Clearly some fraction is removed by the kieselguhr which has no effect on carbon dioxide formation but results in a greater total acetylmethylcarbinol formation. Re]erences p. 556.
VOL.
8 (1952)
ENZYMIC FORMATION OF ACETYLMETHYLCARBINOL
547
Fractionation o/ the enzyme system by precipitation The enzyme system was precipitated without loss of activity by the following method for which acetone dried powders were suitable only if freshly prepared. The powder was extracted with M/I5 phosphate buffer (pH 5.9) at o°C for lO-12 hours and the cell debris removed by centrifugation at low temperature (-2 ° C). The cell free extract was held in a freezing mixture at o ° C and acetone (previously cooled to -15 ° C) added drop by drop with constant stirring to a series of samples of the extract. As soon as sufficient acetone had been added to prevent the extract freezing, the temperature was lowered to - 8 to -IO ° C. The samples of precipitated protein corresponding to known additions of acetone, o-45%, 0-5o%, 0-55% . . . o-8o%, were sedimented b y centrifugation and redissolved in equal volumes of M/I5 phosphate buffer (pH 5.9). The acetylmethylcarbinol and carbon dioxide forming activity of each sample was determined, and a determination of Kjeldahl nitrogen made as a measure of protein precipitation. The results shown in Fig. 2 are typical of those obtained. It will be observed that an increase in the concentration of acetone above 60% to 65 % (v/v), etc., caused a small additional precipitation of protein and this coincided with an inhibition of both the carbon dioxide and acetylmethylcarbinol producing activity of the precipitated protein. This could be due to a separate and distinct enzyme substrate competition or to the precipitation of some other inhibitor or, less likely, to enzyme denaturation. When fractionation was achieved by stepwise addition of acetone to a single extract, 0-4o%, 40-45 %, 45-50% etc., the precipitates corresponding to each addition w e r e
to
o~
"~.~
~
i
:
~.s g
!
i u
fO
20
30
40
SO
60
?0
f~O
~o(v/v)Ace?'one added Fig. 2. Fraction curve for precipitation of acetylmethylcaxbinol enzyme system of A. ~ v o g e ~ s with dioxide activity, - e - - e - - e acetylmethylcaxbinol activity and
acetone.-0--0--0-carbon
--~
Relerences p. 36
556.
,~
~
protein precipitation.
548
VOL. 8 (1952)
F, C. HAPPOLD, C. P. SPENCER
separated by centrifugation and redissolved in equal volumes of phosphate buffer. TABLE I CARBON DIOXIDE
AND
ACETONE
ACETYLMETHYLCARBINOL
PRECIPITATED
FRACTIONS
ACTIVITIES
OF E N Z Y M E
OF SEPARATED
COMPLEX
Composition of Reaction Mixtures: 0. 5 ml (io mg/ml) sodium pyruvate 2.0 ml M/I 5 phosphate buffer, p H 5.8 0.2 ml enzyme fraction
Fraction (vol. acetone added) o-35 % 35-40% 4°-45% 45-50% 50-55% 55-60% 6o-70 %
Carbon dioxide (l~M/6omin/o.2 ml)
A cetylmethylcarbinol (l~M/6omin[o.2 ml)
nil 1.4 7.4 nil 0.8 nil nil
nil 0.9 4.9 nil 0. 4 nil nil
Activity tests conducted with these fractions gave results shown in Table I. There are two very unequal zones of activity, one precipitated 0" between 35 and 45 % (v/v) acetone and a second zone of lesser activity between 50-55 %. A partial reconstruction of the whole extract was achieved by additions of equal quantities of the 45-5o%, 50-55%, 55-60% and 60-70% fractions to the 0-45% fraction. The results of t such combinations (Table II) confirm that the 50-55% fraction (almost without activity) cono0 siderably enhances the activity of the 45 % fraction when added to it. These findings are readily o 6c reproducible and have furthermore been obtained with enzyme extracts from pigs heart muscle 2 (HAPPOLD, HULLIN, NOBLE, AND SPENCER28). The progress curves for carbon dioxide production by the whole extract indicate the presence of two carbon dioxide producing steps in the reaction. The usual course of the reaction is an ~ qr...~, q - - o - - - ~ - initial rapid linear production of carbon dioxide 20 40 60 80 100 for about 3 ° minutes followed by a sharp break T/me in min~ and a subsequent linear period in which the Fig. 3- Progress curves for carbon" dioxide production by the acetone precipitated carbon dioxide production rate is markedly de fractions of the acetylmethylcarbinol creased. The progress curves for carbon dioxide enzyme system of A. aerogenes. -O--©--©0.2 ml o-450/o fraction, production by the 45% and 5o-55% fractions --(~, | ) - - ~ - - 0 . 2 ml 5o-55% fraction alone and when in combination (Fig. 3) indicate and - e - - e - - o o.2 ml 45% fraction that some separation of these two hypothetical plus 0.2 ml 55% fraction steps is occurring. The stimulating effect of the 55 % fraction on the carbon dioxide production of the 45 % fraction is limited to increasing the initial rate of carbon dioxide production only.
jO'
160
jO"
Sy
A?
Re[erences p. 556.
VOL. 8 (1952)
ENZYMICFORMATION OF ACETYLMETHYLCARBINOL
549
TABLE II CARBON
DIOXIDE
COMBINATIONS
AND
ACI~TYLMNTHYLCARBINOL
OF THE
ACETONE
ACTIVITIES
PRECIPITATBD
OF
FRACTIONS
Composition of Reaction Mixtures: o.5 ml (io mg/ml) sodium pyruvate 1.8 ml M / I 5 phosphate buffer, pH 5.8 0.2 ml 45% #action with additions o[ 0.2 ml o] #action as stated
0-45 % 0-45% 0-45% 0-450/o o-45% 0-45%
Carbon dioxide (ttM/6o min/o.2 ml)
Acetylmethylcarbinol (ttM/6o min/o.2 ml)
4.5 4.9 6.9 7.2 6.4 6.5
2.9 3.2 3.4 3.6 3.2 3.2
alone + 45-50% + 5°-55% + 55-60% + 6o-65% + 65-70%
Fresh M/I 5 phosphate buffer (pH 5.9) extract of acetone dried A. aerogenes (volume io ml) Added to
acetone 45%
PRECIPITATE Redissolved in Io ml buffer. 2.0 ml retained (Ist FRACTION
SUPERNATANT (discarded)
To remainder Added to
acetone 45%
PRECIPITATE Redissolved in 8 ml buffer. 2.0 ml retained (2nd FRACTION)
SUPERNATANT (discarded)
To remainder Added to
acetone 45%
PRECIPITATE SUPERNATANT Redissolved in 6 ml buffer. (discarded) 2.o ml used for activity test (3rd FRACTION) Fig. 4- Scheme for fractionation and further separation of the 45% acetone precipitated fraction of the acetylmethylcarbinol enzyme system of A. aerogenes. T h e 45% fraction was f u r t h e r s e p a r a t e d b y s u b m i t t i n g it to t h e f r a c t i o n a t i o n scheme shown in Fig. 4. T h e a c t i v i t y figures for the fractions t o g e t h e r w i t h t h e r e l a t i v e a m o u n t s of p y r u v a t e u t i l i z e d (Table III) were o b t a i n e d a t the end of the reactions as i n d i c a t e d b y a cessation of c a r b o n d i o x i d e e v o l u t i o n (14o minutes). R e p e a t e d p r e c i p i t a t i o n of t h e fraction leads to a m a r k e d decrease in p y r u v a t e utilization, a c e t y l m e t h y l c a r b i n o l a n d c a r b o n d i o x i d e f o r m a t i o n ; it was found impossible to r e p r e c i p i t a t e t h e f r a c t i o n m o r e t h a n twice a n d still r e t a i n a p p r e c i a b l e a c t i v i t y . The m o l a r r a t i o b e t w e e n the carbon dioxide and acetylmethylcarbinol produced remained constant but pyruvate u t i l i z a t i o n does n o t decrease a t t h e same rate, w i t h the result t h a t t h e m o l a r r a t i o p y r u v a t e u t i l i z e d : c a r b o n d i o x i d e p r o d u c e d , increases as follows: whole e n z y m e 1.5; References p. 556.
55 °
F.C.
HAPPOLD,
C. P . S P E N C E R
VOL. 8
(1952)
Ist fraction, 1.8, 2nd fraction, 3.2. Reprecipitation of the 45 % fraction results in the concentration of a p y r u v a t e utilizing enzyme which does not form acetylmethylcarbinol or carbon dioxide. TABLE III A C T I V I T I E S OF T H E FRACTIONS OBTAINED ON F U R T H E R S E P A R A T I O N OF T H E ACETONE P R E C I P I T A T E D FRACTION
45%
Fraction
Pyruvate utilization (l~M#4 o min/I.o ml)
Carbon dioxide production (t~M/I4Omin/I.O ml)
A cetylmethylcarbinol production (#M#4o min/i.o ml)
17.2 14.o 5.8 o.i
i 1.5 7.6 1.8 nil
5.6 3.9 I. I nil
Whole enzyme ist. fraction 2nd. fraction 3rd. fraction
Carbon balance studies with the enzyme system
The results obtained in the previous sections had frequently shown a variation in the theoretical molar ratio of 2 :I for the carbon dioxide and acetylmethylearbinol formed at various times during the course of the reaction. I n addition the fractionation studies had shown the possibility of disproportionate p y r u v a t e utilization. SILVERmAN AND WERKMAN.2 using large scale reaction mixtures had been able to demonstrate a molar ratio of 2 : 2 : 1 for p y r u v a t e utilized, carbon dioxide and acetylmethylcarbinol produced at the end of the reaction. A serial analysis of carbon dioxide and acetylmethylcarbinol production during the course of the enzymic reactions with the unfractionated extracts gave the results shown in Table IV. The theoretical ratio of 2 : 1 for carbon dioxide and acetylmethylearbinol is only reached after about three hours incubation when the reaction was in fact complete; i.e. the acetylmethylcarbinol formation lags behind t h a t of the carbon dioxide during the course of the reaction. The reverse effect is apparent with the o-45 % fraction (Fig. 2 and Table I I ) ; this fraction when isolated gives carbon dioxide acetylmethylcarbinol ratios of 1.5 :I, 1.5 :I and 1.4:1 after 6o minutes incubation, when the 5 o - 5 5 % fraction is present in addition to the 45 % fraction, the observed ratio then becomes 2.o: i and 1.9:1 and thus approximates to the theoretical. TABLE IV SERIAL ANALYSIS OF CARBON D I O X I D E AND A C E T Y L M E T H Y L C A R B I N O L P R O D U C T I O N D U R I N G T H E COURSE OF T H E ENZYMIC REACTION
Composition of Reaction Mixtures: 0.5 ml (5.0 mg/ml) sodium pyruvate 1.7 ml M/I 5 phosphate buffer, pH 5.9 0.5 ml enzyme extract Time (in minutes)
3° 60 ioo 14o i8o References p. 556.
Carbon dioxide A eetylmethylcarbinol produced produced (pM) (l~M)
3.8 5.6 6.5 6.7 6. 7
1.5 2.1 2.9 3.2 3.3
Molar ratio CO2:A MC
2.54 2.67 2.24 2.1o 2.03
VOL. 8 (1952)
ENZYMICFORMATION OF ACETYLMETHYLCARBINOL
551
Possible intermediates in the enzymic reaction T h e t h r e e c o m p o u n d s m o s t likely to occur as i n t e r m e d i a t e s seemed to be acetaldehyde, lactic a n d acetic acids. T h e w o r k of WERKMAN 22 a n d his associates showed t h a t a c e t a l d e h y d e as such was n o t i n v o l v e d in t h e reaction. L a c t i c acid can n o t be d e t e c t e d in s a m p l e s from large scale r e a c t i o n m i x t u r e s a t a n y t i m e d u r i n g t h e r e a c t i o n b y either the t h i o p h e n e r e a c t i o n of HOrKINS or UFFELMANN'S reagent. Similarly, a f t e r r e m o v i n g interfering ions b y the p r o c e d u r e of HUTCHENS AND KASS2s n e g a t i v e results are o b t a i n e d in t h e l a n t h a n u m n i t r a t e t e s t for acetate. No volatile acids can be d e t e c t e d in t h e distillate after r e m o v a l of p y r u v i c acid b y redistillation from an acid m a g n e s i u m mercuric sulphate mixture. T h e effect of b o t h a c e t a l d e h y d e acetic a n d lactic acids on 240 10) f t h e e n z y m i c r e a c t i o n was investig a t e d . T h e results confirm t h e 2O0 O f findings of SILVEI~MAN AND I-O--O ....-0/ WERKMAN t h a t acetaldehyde f does n o t e n t e r into t h e reaction. ~ L a c t a t e is w i t h o u t effect e i t h e r _~ u p o n t h e r a t e s of f o r m a t i o n or ._~ 120 final a m o u n t s of c a r b o n dioxide and acetylmethylcarbinol formed ~ or u p o n t h e a m o u n t of p y r u v a t e Q. ~ 86 utilized, in a d d i t i o n t h e e n z y m e s y s t e m is w i t h o u t effect u p o n 40 ' l a c t a t e alone. A c e t a t e , on t h e other hand, stimulates both the 0 20 40 60 80 100 q20 r a t e a n d a m o u n t of carbon diTime m rain= oxide a n d a c e t y l m e t h y l c a r b i n o l p r o d u c t i o n (Fig. 5 a n d T a b l e V), Fig. 5. The effect on carbon dioxide production of the addition of acetate to pyruvate dissimilations by the acetyli t s effect on t h e f o r m e r being methylcarbinol enzyme system of A. aerogenes. l i m i t e d to increasing t h e initial -O--O~O23/~M pyruvate, r a t e of c a r b o n d i o x i d e f o r m a t i o n . -~, , - - e - 23 p M pyruvate plus 2o/~M acetate.
.o//
TABLE V EFFECT
OF ADDITIONS
AMOUNTS
OF ACETATE
OF CARBON DIOXIDE
TO P Y R U V A T E AND
DISSIMILATIONS
ACETYLMETHYLCARBINOL
ON T H E
TOTAL
FORMED
Composition of Reaction Mixtures: 0. 5 ml (5.0 mg/ml) sodium pyruvate o. 5 ml enzyme extract 1.2 ml M/I5 phosphate buffer, pH 5.9
Addendum 0. 5 ml phosphate 0. 5 ml phosphate 0. 5 ml phosphate 0. 5 ml phosphate o. 5 ml phosphate 0. 5 ml phosphate
References P. 556.
buffer buffer buffer buffer + 0. 5 ml (20 pM/ml) acetate buffer + o. 5 ml (2o pM/ml) acetate buffer + o. 5 ml (20 pM/ml) acetate
Carbon dioxide produced (pM)
A cetylmethylcarbinol produced (aM)
7.6 7.2 7.3 8.9 9.0 9.2
3.7 3.6 3.7 4.2 4.2 4.5
552
VOL. 8
F . C . HAPPOLD, C. P. SPENCER
(I952)
No carbon dioxide or acetylmethylcarbinol are formed by the system from acetate alone. The "catalytic" effect of acetate on the enzymic reaction was further demonstrated by an examination of the effect of acetate concentration on the magnitude of the stimulation produced. The results shown in Fig. 6 are typical of those obtained. With the sample of the extract used, as little as 2/~M of acetate produces a maximum effect. Above this concentration the magnitude of the stimulation is largely independent of the acetate concentration though an additional stimulation is invariably noticed when the molar acetate concentration approaches that of the pyruvate.
I
100
o
Q.
j
L
•~ 8.O(
a ~ O
°) ~u
~
2.0
~'
o
5.0
10.0 150 200 Acetate added in ~1~
I
2riO
300
Fig. 6. The effect of concentration of acetate on carbon dioxide - O - - O - - O and acetylmethylcarbinol production - e - - e - - e by enzyme system from A. aerogenes during dissimilation of 2 3/~M of pyruvate.
DISCUSSION
Since these studies were completed JuNI 3° briefly reports a more complete fractionation of the enzyme from A. aerogenes. Of the two fractions he obtained, one acts on pyruvic acid to form a-acetolactic acid and carbon dioxide and the other decarboxylates this to yield acetylmethylcarbinol and carbon dioxide, the latter is without effect upon pyruvic acid. In ~:ddition, a variety of Vosges Proskauer positive organisms and the acetylmethylcarbinol enzyme system from pigs heart have been shown to be able to decarboxylate a-acetolactic acid (WATT AND KRAMPITZ3Z; KRAMPITZ AND EVERETTa*) whilst organisms which do not ordinarily produce acetylmethylcarbinol are capable of catalysing this reaction. The first "pyruvate decarboxylase" fraction has been shown to be cocarboxylase dependent whilst the a-acetolactic acid decarboxylase system is dependent upon manganese or magnesium ions but not cocarboxylase. This work leaves little doubt that the synthesis of acetylmethylcarbinol can occur through a-acetolactic acid as an intermediate. OCHOA3~ is of the opinion that the mechanism of acetylmethylcarbinol synthesis is best explained by the following reactions: Re/erences p. 556.
VOL. 8 (1952)
ENZYMIC FORMATION OF ACETYLMETHYLCARBINOL
a. Pyruvic acid + Enzyme
COs + Acetylmethylcarbinol ~ b. Pyruvic acid + Enzyme
,
>
553
(Acetaldehyde-enzyme) + CO2 + Pyruvic Acid Acetolactic acid + Enzyme
> (Acetaldehyde-enzyme) + COn Acetaldehyde + Enzyme + (Acetaldehyde-enzyme) Acetylmethylcarbinol + Enzyme
There is evidence against the occurrence of acetaldehyde as an intermediate in the bacterial enzymic synthesis of acetylmethylcarbinol and the formation of an enzyme complex is postulated to explain this. The second mechanism is postulated to account for the condensation of added acetaldehyde by the system from yeast and animal tissues, a-acetolactic acid not occurring as an intermediate in this scheme. The results reported in this paper confirm much of the findings reviewed above. We have shown that the whole enzyme system has a resultant sharp optimum pH at about 5.9 for both acetylmethylcarbinol and carbon dioxide formation. This is in agreement with the original results of SILVERMAN AND WERKMAN22 who stated that the pH optimum for the system was in the region of 5.6 to 6.0. The results obtained by these workers were irregular and some of their preparations were apparently as active at pH 4.5 as at 5.6. Our preparations were all comparatively inactive at a pH of 4.5, considerable protein precipitation occurring below this pH. Use of half saturated potassium chloride as an extracting agent for the system and the kieselguhr effect both indicated the presence of two enzymic fractions. Similarly the relative effects of coenzyme factors and metallic ions on acetylmethylcarbinol and carbon dioxide production are in accord with the presence of two distinct systems, one cocarboxylase dependent and one manganese dependent. The partial fractionation achieved indicated the presence of a pyruvate utilizing fraction and of a second fraction that was inactive towards pyruvate in the absence of tile first fraction. It seems most probable that our second fraction is identical with the a-acetolactic decarboxylase of J u s I 8°. The kinetics of carbon dioxide production by the whole system are in agreement with the conception of two distinct decarboxylating fractions, the first being associated with the initial attack on pyruvate and the second with the a-acetolactic decarboxylase. Furthermore it appears that the decarboxylating activity of the second fraction is conditional upon prior decarboxylation of the pyruvate by a system contained in the first fraction. In contrast, however, further attempted purification of our first fraction indicates that a "pyruvate utilizing non-carbon dioxide forming" system may be present which suggests that JuNI'S first fraction may not be a single enzyme. Serial analysis of the products during the course of the reaction indicates that the theoretical molar ratio of 2 : I for carbon dioxide and acetylmethylcarhinol produced is only obtained at the end of the reaction. In addition it has been shown that acetate (but not acetaldehyde) is condensed by the bacterial enzyme system during the concurrent decarboxylation of pyruvate; and causes an increase in both the rate and amount of formation of carbon dioxide and acetylmethylcarbinol. These results are not entirely compatible with the reaction paths suggested by OCI~OA33. Hydrogen acceptors have long been known to act on the enzymic formation of Re~erences p. 556.
554
F.C. HAPPOLD, C. P. SPENCER
VOL. 8
(1952)
acetylmethylcarbinol by increasing (or in the case of yeast, initiating) its formation, and it has been supposed that they exerted an "acetaldehyde sparing" action thus providing more of the two-carbon compound for condensation to acetylmethylcarbinol. Studies with washed cell suspensions of A. aerogenes (HAPPOLD AND SPENCER26) have indicated that an oxidative reaction may be involved in the conversion of pyruvic acid to acetylmethylcarbinol and that such an oxidative reaction m a y be linked with a reductive reaction (other than the reduction of the carbinol to the glycol) which also occurs in the reaction chain. An added hydrogen donor or a hydrogen donating reaction of the glycolytic cycle is able to act in place of the normal hydrogen donating system. MARTIUS~4 has proposed a hypothesis to explain pyruvate metabolism in which he postulates that the first reaction of pyruvate in the presence of co-carboxylase is dehydrogenation, and some evidence has been presented by SOUMALINER AND JXNNES~5 that dehydrogenation is the primary phase of pyruvate breakdown prior to acetylmethylcarbinol formation by yeast. I t therefore seems possible that an oxidative decarboxylation of pyruvate occurs and that acetate (or a compound closely related to it other than acetaldehyde) is the two-carbon compound involved in acetylmethylcarbinol formation. In this case, the absence of an acetaldehyde effect with the bacterial enzyme extract m a y be due to the absence of an aldehyde oxidase. In contrast, with extracts from mammalian tissue acetaldehyde m a y have a dual role (i.e. as a source of supply of the two-carbon compound and as a hydrogen donor). This would account for the fact that although the acetate effect can be observed with mammalian enzyme extracts, the analogous acetaldehyde effect is markedly greater. If acetate is involved two possibilities arise, a-acetolactic acid could arise by interaction of acetate and lactic acid. Such a scheme would involve an oxidative decarboxylation and a reduction of pyruvate and might account for the observed effects of hydrogen donors and acceptors on the system. The fact that lactate is not condensed by the bacterial system m a y be due to its not appearing as such in the reaction (enzyme complex?). Alternatively, a condensation between acetate and acetaldehyde (as an enzyme complex) is possible: CHs I CO I OH H q CO CH a
CH 8
CH a
I
--H20
CO I * CO CH a
I
+
2H
CH.0H ~ CO CH a
-
A reaction of this nature might pass through diacetyl as an intermediate which would provide the symmetrical intermediate that the isotopic studies indicate. There is no direct evidence for the occurrence of diacetyl in this role but SOUMALINER AND JXNNES3s claim to have detected diacetyl in yeast dissimilations of pyruvic acid under conditions that make its formation by fortuitous oxidation of the carbinol unlikely. In addition, an enzyme system has been isolated from mammalian tissue which catalyses the reduction of diacetyl to acetylmethylcarbinol (STuMPF, GREEN AND ZARUDNAYA36~. The isolated system uses a second molecule of diacetyl as a hydrogen donor but it is possible that, if this system is involved in the enzymic synthesis of acetylmethylcarbinol, it might be linked to the oxidative decarboxylation of pyruvate. Previous work with
Relerences p. 556.
VOL. $
(1952)
ENZYMICFORMATION OF ACETYLMETHYLCARBINOL
555
aldehydes (NEUBERG AND HIRSCH1; BERG AND WESTERFIELD 37) m a k e s i t appear probable that the condensation of acetaldehyde involves the coupling of the aldehyde (or its derivative) with a two-carbon compound and not with pyruvate or lactate, in which case a-acetolactic acid may not be involved in this pathway. The bulk of the evidence at the present time seems to indicate that this is the case, the kinetic data for carbon dioxide production on additions of acetate indicating that the increased rate involves the decarboxylation associated with the first fraction and not the aacetolactic decarboxylase fraction. To explain the increased rate of carbon dioxide formation on addition of acetate it is necessary to assume that the condensation between pyruvate and the acetaldehyde-enzyme complex is the rate limiting step, added acetate thus being able to compete with the excess pyruvate. OCHOA'S scheme for the intervention of acetaldehyde (reaction scheme b) may be redrafted as below: substituted
Pyruvic acid + Enzyme Pyruvic acid
) (Acetaldehyde-enzyme) + CO S
+ 1~ O3 ) Acetate + CO s
(Acetaldehyde-enzyme) + Acetate
Acetaldehyde
[
> Diacetyl + Enzyme
I 2H Acetylmethylcaxbinol
Such a modification provides an explanation of the effects of acetate and of hydrogen donors on the reaction. It does not appear that this mechanism operates through a-acetolactic acid as an intermediate and hence it must be supposed to function as an alternative and in addition to that reaction path. The authors wish to thank the Medical Research Council for a grant made in support of this work. SUMMARY A cell-free enzyme extract which catalyses the anaerobic decaxboxylation of pyruvic acid with the formation of acetylmethylcarbinol has been isolated from Aerobacter aerogenes b y three methods (viz. b y grinding with powdered glass, b y shaking with glass beads and b y extraction of a n acetone dried preparation). Adsorption of the complex, fractionation studies and an examination of the effects of coenzyme factors indicate t h a t a t least two systems are involved. The whole system is able to condense acetate b u t not acetaldehyde during the concurrent decarboxylation of pyruvate. I t is suggested t h a t acetate m a y be involved in one type of enzymic synthesis of acetylmethylcarbinol and t h a t acetaldehyde is condensed only after oxidation to acetate. The possibility of the occurrence of diacetyl as an intermediate in the reaction involving acetate is discussed. R~SUM]~ Un suc enzymatique d6pourvu de cellules agissant comme catalisateur de la d6carboxylation ana6robique de l'acide pyruvique avec la production d'acdtylmethylcarbinol a 6t6 obtenu de l'Aerobaaer aerogenes au moyen de trois m~thodes: en b r o y a n t les bact6ries avec du verre pulveris~, en les agitant avec des billes de verre, ou enfin par l'extraction d'une preparation sech~e avec de l'acdtone. Les donn~es fonrnies: par l'adsorption de ce suc enzymique, par les r~sultats du fractionnement et l'effet des agents activateurs indiquent qu'au moins deux syst~mes enzymatiques y sont present. L'ensemble est capable de condenser l'acdtate mais non pas l'ac6taldehyde lors de la d~carboxylation simultan~e du pyruvate. On peur supposer que l'ac~tate intervient dans un genre de synth~se enzymatique d'acdtylmethylcarbinol et que l'acdtaldehyde est condens6 seulement apr~s avoir 6t6 transform6 en ac$tate par oxydation. La possibilit6 de la presence du diacetyl comme interm6diaire dans cette rdaction est discut~e.
Re/erences p. 556.
556
F . C . HAPPOLD, C; P. SPENCER
VOL. 8
(1952)
ZUSAMMENFASSUNG E i n zellenfreier E n z y m s a f t , welcher die a n a e r o b e D e c a r b o x y l a t i o n der B r e n z t r a u b e n s A u r e u n t e r B i l d u n g v o n Acetoin katalisiert, w u r d e a u s d e m Aerobacter aerogenes a u f d r e i f a c h e m Wege, n~kmlich d u t c h ZerTeiben m i t p u l v e r i s i e r t e m Glass, d u r c h Schfitteln m i t Glassperlen u n d d u t c h E x t r a h i e r e n eines m i t A c e t o n g e t r o c k n e t e n P r ~ p a r a t e s e r h a l t e n . Die A d s o r p t i o n des E n z y m s a f f e s , die E r g e b n i s s e der F r a k t i o n i e r u n g , sowie die B e o b a c h t u n g e n iiber die W i r k u n g der A k t i v a t o r e n weisen d a r a u f hin, d a s s w e n i g s t e n s zwei E n z y m s y s t e m e dabei beteiligt sind. D a s S y s t e m als G a n z e s ist i m s t a n d e A c e t a t , n i c h t a b e r A c e t a l d e h y d wXhrend der gleichzeitigen D e c a r b o x y l a t i o n der B r e n z t r a u b e n s ~ u r e zu k o n d e n s i e r e n . Es wird a n g e n o m m e n , d a s s d a s A c e t a t in einer der e n z y m a t i s c h e n S y n t h e s e n des Acetoins beteiligt sein k 6 n n t e u n d d a s s A c e t a l d e h y d erst n a c h O x y d a t i o n zu A c e t a t k o n d e n s i e r t wird. Die M6glichkeit des A u f f r e t e n s v o n D i a c e t y l als einer Z w i s c h e n s t u f e in dieser R e a k t i o n wird erOrtert. REFERENCES 1 c. NEUBERG AND J. HIRSCH, Biochem. Z., 115 (1921) 282. 2 C. N~UBERG AND L. LEIB]~RMANN, Biochem. Z., 121 (1921) 311, 3 C. NEUBERG AND H. OHLE, Biochem. Z., 127 (1922) 327 . C. NEUBERG AND H. OHLE, Biochem. Z., 128 (1922) 61o. s C. NEUBERG AND E. REINFURTH, Biochem. Z., 143 (1923) 553. e C. NEUBERG AND A. MAY, Biochem. Z., 14o (1923) 299. 7 C. NEUBERG AND O. ROSENTHAL, Ber., 5 7 B (1924) 1436. s A. J. KLUYVER AND H. J. L. DONKER, Proc. Acad. Sci. Amsterdam, 28 (1924) 314 . 9 y . TOMIYASU, Biochem. Z., 289 (I936) 9710 y . TOMIYASU, Rept. Japan Assoc. Advancement Sci., 16 (1942) 552. 11 A. KuzlN, Chemical Abstracts, 31 (1937) 5386/9. 11 W. DIRSCHERL, Z. physiol. Chem., 188 (193o) 225. 13 W. DIRSCHERL, Z. physiol. Chem., 252 (1938) 7 o. 14 B. TANKO AND L. MUNK, Z. physiol. Chem., 262 (1939) 144. 15 N. H. GROSS AND C. H. WERKMAN, Antonie van Leeuwenhoeck. J. Microbiol. Serol., 12 (1947) 19. 16 N. H. GROSS AND C. H. WERKMAN, Arch. Biochem., 15 (1947) 125. 17 D. E. GREEN, W . W. W~STERFIELD, B. VENNESLAND, AND W . E. KNOX, J . Biol. Chem., 145 (1942 ) 69. is M. M. BARRITT, J . Path. Bact., 44 (1937) 679. 19 M. N. MICHELSON AND C. H. WERKMAN,J, Bact. 37 (1939) 619., 20 H. REYNOLDS, B. J. JACOBSSON, AND C. H. WERKMAN, J. Bact., 34 (1937) 15. 21 H. G. STAHYL AND C. H. WERKMAN, Biochem. J., 36 (1942) 575. $2 N. SILVERMAN AND C. H. WERKMAN, J. Biol. Chem., 138 (1941) 35. 23 H. TAUBER, J. Biol. Chem., 125 (1938) 191. F. C. HAPPOLD AND C. P. SPENCER, Biochim. Biophys. Acta, in t h e press. 35 T. E. FRIEDMANN AND G. E. HAUGEN, J. Biol. Chem., 147 (1943) 415 • R. MARKHAM, Biochem. J., 36 (1942) 790. 27 E. F, GA.LE, The Chemical Activities o/Bacteria, (1947). U n i v e r s i t y T u t o r i a l Press, L o n d o n . 3s F. C. HAPPOLD, R. P. HULLIN, R. L, NOBLE, AND C. P. SPENCER, (unpublished). 29 j . O. FIUTCHENS AND B. M. KASS, J . Biol. Chem., 177 (1949) 571. 30 E. JUNI, Federation Proc., 9 (195 o) 396. 31 D. WATT AND L. O. KRAMPITZ, Federation Proc., 6 (1947) 3 ol. 33 L. O. KRAMPITZ AND J. EVERETT, Arch. Biochem., 17 (1948) 8I. 33 S. OCHOA, Physiol. Rev., 31 (1951) 56. 34 C. MARTIUS, Z. physiol. Chem., 279 (1943) 96. 35 H. SUOMALAINEN AND L. JANNES, Nature, 157 (1946) 336. as p. K. STUMPF, D. E. GREEN, AND K. ZARUDNAYA, J. Biol. Chem., I67 (1947) 811. 37 R. L. BERG AND W . W . WESTERFIELD, J. Biol. Chem., 152 (1944) xi3Received,
June
i6th,
1951
BIOCHIMICA ET BIOPHYSICA ACTA
543
T H E ENZYMIC FORMATION OF ACETYLMETHYLCARBINOL AND R E L A T E D COMPOUNDS by FRANK C. HAPPOLD AND C. P. SPENCER
Department o/Biochemistry, University o/Leeds (England)
INTRODUCTION
Acetylmethylcarbinol, 2:3 butylene glycol and diacetyl are of widespread occurrence as products of carbohydrate metabolism. The problem of the mechanism of their formation has attracted numerous workers, the first investigations being made by NEUBERG and his associates working with yeast1, 2, 3,4, 5, s, 7. They suggested that pyruvic acid was first decarboxylated (by carboxylase) to acetaldehyde which then condensed to form acetylmethylcarbinol. They postulated an enzyme "carboligase" to catalyse this reaction. Although acetylmethylcarbinol is not formed during the normal fermentation of glucose by yeast, additions of acetaldehyde (or other hydrogen acceptors) cause its formation. The anomaly that the carbinol is not formed from glucose alone was later suggested by KLUYVERAND DUNKERs to be due to the rapid removal of acetaldehyde by reduction. No satisfactory biosynthesis of acetylmethylcarbinol using acetaldehyde alone was achieved although subsequently several reports of such a reaction have appeared (ToMIYASU9,10 and KUZlNn). DIRSCHERL12,13 has suggested that the carbinol is formed by the non-enzymic condensation of active (i.e. biologically produced) acetaldehyde, a hypothesis that gained support by the failure of several workers to demonstrate a separate carboxylase and carboligase (TANKO AND MUNK14). Isotopic studies have since confirmed that acetaldehyde added to yeast fermentations is converted to acetylmethylcarbinol but they did little to elucidate the actual mechanism of its conversion (GROSSAND WERKMAN TM16). The data obtained from enzyme studies with mammalian tissue extracts are in many ways analogous to those obtained during studies with yeast. The addition of acetaldehyde to the decarboxylation of pyruvic acid by an enzyme system isolated from pigs heart causes a marked increase in the rate of both carbon dioxide and acetylmethylcarbinol production (GREEN, WESTERFIELD, VENNESLAND, AND KNOX17), and both systems are able to form acetylmethylcarbinol from acetaldehyde alone. Somewhat conflicting data have been obtained in studies with bacteria. Pyruvic acid acts as a precursor of both carbinol and glycol (BARRITTTM) and the addition of acetaldehyde (MICHELSON AND WERKMAN 19) or acetic acid (REYNOLDS AND WERKMAN 20) to fermentations of glucose by Aerobacter indologenes and Bacillus polymyxa (STAHYL AND WERKMANll) cause an increased formation of the four-carbon compounds. The position was further complicated by the preparation of a cell free enzyme system from A. aerogenes which catalyses the anaerobic decarboxylation of pyruvic acid with concurrent formation of acetylmethylcarbinol (SILVERMAN AND WERKMAN22), but does not Re#rences p. 556.
544
v . c . HAPPOLD, C. P. SPENCER
VOL. 8
(1952)
utilize acetaldehyde or acetyl phosphate during the dissimilation of pyruvate. Later isotopic studies (GRoss AND WERKMAN 15) confirmed these results and indicated that at least two mechanisms existed. It was suggested that whilst the condensation of acetaldehyde can be accomplished by whole cells this system is absent from cell-free extracts which produce aeetylmethylcarbinol by another mechanism. The studies reported in this paper are primarily concerned with the mechanism of formation of acetylmethylcarbinol by bacteria and we have endeavoured to relate the results obtained with those from studies with other systems. EXPERIMENTAL METHODS
Materials The sample of co-carboxylase used was synthesised by the method of TAUBER*S; sodium p y r u v a t e was prepared as previously described (HAPPOLD AND SPENCER~a).
Methods o] Estimation Pyruvie acid was determined colorimetrically as the 2:4 dinitrophenylhydrazone following a specific extraction procedure (FRIED~MANN AND HAUGEN~). Protein nitrogen was e s t i m a t e d b y a micro-Kjeldahl m e t h o d (MARKHAM'% Acetylmethylcarbinol was determined b y the m e t h o d described in a previous paper 14. No 2 : 3 butylene glycol was formed b y t h e system. Carbon dioxide was measured by conventional WARBURGtechnique using air as the gas phase in the flasks. Preliminary extmrim~nts showed t h a t carbon dioxide evolution was t h e only gas exchange catalysed b y the crude enzyme, oxygen uptake being zero or negligible. Carbon dioxide evolution was measured at 37 ° C, p y r u v a t e being added from the side a r m a t zero time. The m a i n chamber of the flasks n o r m a l l y contained t h e enzyme solution a n d other a d d e n d a in M[I 5 p h o s p h a t e buffer at p H 5.9. I n t h e experiments when parallel estimations of carbon dioxide and acetylmethylcarbinol were made, the carbon dioxide production was first e s t i m a t e d m a n o m e t r i c a l l y a n d immediately afterwards samples were withdrawn from t h e flasks, pipetted into a protein precipitant ( n o r m a l l y lO% trichloracetic acid) and t h e acetylmethylcarbinol determined. Parallel control experiments using a boiled sample of t h e enzyme a n d w i t h o u t addition of the s u b s t r a t e were always performed.
Growth o] Cells A. aerogenes (laboratory strain) was m a i n t a i n e d b y fortnightly sub-culturing on ordinary agar slopes. The organism was grown in bulk in io 1 quantities of the following m e d i u m : I . o % glucose, 0 . 3 % peptone, o.8% dipotassium hydrogen phosphate, i o % t a p water (metals). The t a p water and p h o s p h a t e were autoclaved separately and mixed asceptically after sterilization. The bulk m e d i u m was inoculated with t h e whole of t h e growth from a 24 hour slope culture of the organism a n d was incubated for 2o to 24 hours a t 37 ~ C. The cells were harvested in the form of a paste b y m e a n s of an Alfa Laval Centrifugal Oil Separator.
Preparation o] Cell-Free Enzyme Extracts SILVERMAN AND WERKMAN22 ground their cells with powdered glass in preparing their e x t r a c t s whilst GALE~7 shook cell suspensions with glass beads to liberate the acetylmethylcarbinol forming s y s t e m from the bacteria. Both m e t h o d s have been used in t h e present work. Active e x t r a c t s were also obtained from acetone dried bacteria prepared b y the following m e t h o d which overcomes the difficulties of drying ceils producing mucilagenous polysaccharide in their growth. The cells were grown in the usual glucose-peptone media adjusted to p H 6.0 before inoculation. The bulk m e d i u m was inoculated by transferring a loop of growth from a 24 h o u r agar slope culture of the organism to 9 ml of sterile water and agitating until the suspension became j u s t observably turbid. The bulk m e d i u m was inoculated with the whole of t h i s suspension a n d was incubated at 3 °° C for a m a x i m u m of 2o hours. These details resulted in somewhat impaired yields b u t limited the a m o u n t of polysaccharide formed by t h e cells a n d so minimised their resistance to the action of t h e acetone. Failure to observe these details always resulted in a yield of cells unsuitable for acetone drying. The cells from IO 1 of m e d i u m were harvested, washed, resuspended in 2oo ml of distilled water a n d cooled to o ° C. The chilled suspension was t h e n slowly added to 5 volumes of ice cold acetone, t h e addition being made slowly and with c o n s t a n t agitation; the suspension was stored at - 1 5 ° C for 15 to 24 hours. The cells were finally separated by filtration, washed first with 75 ° ml of 50% acetone-ether and finally with 250 ml of ether, care being taken to see t h a t the powder did not become dry until after t h e final washing with ether. Final drying was t h e n accomplished b y suction of air t h r o u g h the .filter.
Re#fences p. 556.
VOL.
8 (1952)
ENZYMIC FORMATION OF ACETYLMETHYLCARBINOL
545
A c e t o n e - d r i e d p o w d e r s of A. aerogenes p r e p a r e d in t h i s w a y were q u i t e stable if s t o r e d in a d e s i c c a t o r a t r o o m t e m p e r a t u r e . T h e e n z y m e s y s t e m w a s e x t r a c t e d w i t h M / I 5 p h o s p h a t e buffer, p H 6.0, t h e o p t i m u m t i m e of e x t r a c t i o n being 12 h o u r s a t o ° C. T h e cell debris w a s s e d i m e n t e d b y c e n t r i f u g a t i o n for 3 ° to 45 m i n u t e s a t 7ooo r.p.m. T h e a v e r a g e yield of cell free e x t r a c t f r o m io 1 of m e d i u m w a s a b o u t 15 ml.
General E n z y m e e x t r a c t s o b t a i n e d b y all t h r e e m e t h o d s q u i c k l y lost a c t i v i t y , five h o u r s s t a n d i n g a t r o o m t e m p e r a t u r e b e i n g sufficient to c a u s e i n a c t i v a t i o n . Irreversible i n a c t i v a t i o n occurred after a b o u t 3 ~ h o u r s a t 37 ° C e v e n in t h e presence of t h e s u b s t r a t e , a n d a t o ° C c o m p l e t e i n a c t i v a t i o n occurred a f t e r 3o hours. A d d i t i o n s of i n e r t proteins, p r o t e c t i v e colloids a n d r e d u c i n g a g e n t s did n o t stabilize t h e s y s t e m . T h e cell free e x t r a c t s were, therefore, stored in a frozen c o n d i t i o n a t - ! 5 ° C. SILVERMAN AND WERKMAN r e p o r t e d t h a t if frozen solid, e x t r a c t s could be s t o r e d for a considerable period w i t h l i t t l e loss of d e c a r b o x y l a t i n g a c t i v i t y . O u r experience h a s confirmed t h i s b u t h a s also s h o w n t h a t freezing a n d t h a w i n g are n o t w i t h o u t effect u p o n t h e a c e t y l m e t h y l e a r b i n o l a c t i v i t y . T h e practice of s t o r i n g e x t r a c t s before use was, therefore, avoided. EXPERIMENTAL
RESULTS
Determination o/the optimum p H o/the enzyme system A sample of the cell free extract was obtained 'by the glass grinding technique. The production of carbon dioxide and acetylmethylcarbinol from pyruvate by the enzyme preparation was measured in citrate-phosphate buffers at various pH's determined by a Glass Electrode. There was no appreciable change in the p H of any of the reactants during incubation. The results shown in Fig. I are typical of those obtained. The determinations of the optimum p H of carbon dioxide and acetylmethylcarbinol were made on different samples of the extract and the relative amounts of the two compounds formed are not comparable. The p H optimum of the system is about 5.9 for both acetylmethylcarbinol and carbon dioxide production. All extracts were inactive below a pH of 4 when protein precipitation occurred.
(o}
I
,l
(b)
30o
i
~ 2oo "~150
o
~
3.O
4.0
5.0
60
~o
~oo
3.g
4.0
~0
60
7.0
~0
Fig. I. p H a c t i v i t y c u r v e s for a c e t y l m e t h y l c a r b i n o l e n z y m e of A. aerogenes. (a) a c e t y l m e t h y l c a r b i n o i p r o d u c t i o n . (b) c a r b o n dioxide p r o d u c t i o n .
Re/erences p. 556.
546
F . C . HAPPOLD, C. P. SPENCER
VOL. 8
(1952)
Comparison o[ the cell-/ree extracts obtained by the three methods o] preparation The extracts prepared by the three methods of preparation were compared by means of an arbitrary unit of activity. This was defined as: that amount of acetylmethylcarbinol (in ~g) or carbon dioxide (in ~1) produced by an amount of enzyme containing I mg protein nitrogen after 3 ° minutes incubation at 37 ° C in M/I 5 phosphate buffer at pH 5.9 in the presence of excess pyruvate. The results place the shaking method as the most efficient but the extracts obtained from the acetone dried powders are the most convenient to prepare, and such extracts have been used in all further studies unless it is stated otherwise. In the course of these studies, whilst comparing KC1 with the M/I 5 phosphate as an extraction agent for the enzyme system, it was observed that variations in the strength of the KC1 employed (from 1/s to ~ saturation) caused COs evolution to drop from IiO to 32 /A/3 o min/mg N~, whereas acetylmethylcarbinol production fell only from 290 to 260/~g.
The effect o] co-carboxylase, manganese and magnesium ions on the enzyme system SILVERMAN AND WERKMAN reported that co-carboxylase, manganese and, to a lesser extent, magnesium ions, increase the rate of decarboxylation of pyruvate. These effects were reinvestigated by measuring the effects of these ions on both carbon dioxide and acetylmethylcarbinol production. Saturation of the system with either co-carboxylase or manganese ions stimulates the initial rate of carbon dioxide and of acetylmethylcarbinol production. The observation that magnesium ions acted similarly could not be confirmed. Co-carboxylase alone causes a disproportionate stimulation of the initial rate of carbon dioxide production. In fresh samples of the extract equality in the magnitude of the effect on the two activities is restored by concurrent saturation of the system with manganese ions. It proved impossible to resolve the system by dialysis (with a view to its subsequent reconstruction from known dialysable factors) since, owing to its instability at temperatures above its freezing point, irreversible inactivation of the protein moiety occurs. Dialysable factors are, however, involved since a dialysate (obtained by dialysis of a cell free extract against distilled water) after concentration in a vacuum at 25 ° C shows a stimulating action on the initial rates of both carbon dioxide and acetylmethylcarbinol production.
Adsorption o/the enzyme complex When an active phosphate buffer extract of acetone dried powder is shaken with kieselguhr, the supernatant removed and the kieselguhr washed with phosphate buffer at pH 5.9 an interesting phenomenon occurs. The carbon dioxide activity of the control is 430 ~1/3o min/o.5 ml, the equivalent of the supernatant and the washings 322 and I21 ~1 respectively. Apparently no adsorption takes place and the agreement between the control and the summated fractions is excellent. When aliquots of the same preparations are tested for acetylmethylcarbinol, however, the summated activity of the fractions are apparently greater than the starting material alone, moreover, the acetylmethylcarbinol/carbon dioxide ratio (~M/ml) changes from 1.29 with the control material to 1.63 with the supernatant and 2.1 with the washings. Clearly some fraction is removed by the kieselguhr which has no effect on carbon dioxide formation but results in a greater total acetylmethylcarbinol formation. Re]erences p. 556.
VOL.
8 (1952)
ENZYMIC FORMATION OF ACETYLMETHYLCARBINOL
547
Fractionation o/ the enzyme system by precipitation The enzyme system was precipitated without loss of activity by the following method for which acetone dried powders were suitable only if freshly prepared. The powder was extracted with M/I5 phosphate buffer (pH 5.9) at o°C for lO-12 hours and the cell debris removed by centrifugation at low temperature (-2 ° C). The cell free extract was held in a freezing mixture at o ° C and acetone (previously cooled to -15 ° C) added drop by drop with constant stirring to a series of samples of the extract. As soon as sufficient acetone had been added to prevent the extract freezing, the temperature was lowered to - 8 to -IO ° C. The samples of precipitated protein corresponding to known additions of acetone, o-45%, 0-5o%, 0-55% . . . o-8o%, were sedimented b y centrifugation and redissolved in equal volumes of M/I5 phosphate buffer (pH 5.9). The acetylmethylcarbinol and carbon dioxide forming activity of each sample was determined, and a determination of Kjeldahl nitrogen made as a measure of protein precipitation. The results shown in Fig. 2 are typical of those obtained. It will be observed that an increase in the concentration of acetone above 60% to 65 % (v/v), etc., caused a small additional precipitation of protein and this coincided with an inhibition of both the carbon dioxide and acetylmethylcarbinol producing activity of the precipitated protein. This could be due to a separate and distinct enzyme substrate competition or to the precipitation of some other inhibitor or, less likely, to enzyme denaturation. When fractionation was achieved by stepwise addition of acetone to a single extract, 0-4o%, 40-45 %, 45-50% etc., the precipitates corresponding to each addition w e r e
to
o~
"~.~
~
i
:
~.s g
!
i u
fO
20
30
40
SO
60
?0
f~O
~o(v/v)Ace?'one added Fig. 2. Fraction curve for precipitation of acetylmethylcaxbinol enzyme system of A. ~ v o g e ~ s with dioxide activity, - e - - e - - e acetylmethylcaxbinol activity and
acetone.-0--0--0-carbon
--~
Relerences p. 36
556.
,~
~
protein precipitation.
548
VOL. 8 (1952)
F, C. HAPPOLD, C. P. SPENCER
separated by centrifugation and redissolved in equal volumes of phosphate buffer. TABLE I CARBON DIOXIDE
AND
ACETONE
ACETYLMETHYLCARBINOL
PRECIPITATED
FRACTIONS
ACTIVITIES
OF E N Z Y M E
OF SEPARATED
COMPLEX
Composition of Reaction Mixtures: 0. 5 ml (io mg/ml) sodium pyruvate 2.0 ml M/I 5 phosphate buffer, p H 5.8 0.2 ml enzyme fraction
Fraction (vol. acetone added) o-35 % 35-40% 4°-45% 45-50% 50-55% 55-60% 6o-70 %
Carbon dioxide (l~M/6omin/o.2 ml)
A cetylmethylcarbinol (l~M/6omin[o.2 ml)
nil 1.4 7.4 nil 0.8 nil nil
nil 0.9 4.9 nil 0. 4 nil nil
Activity tests conducted with these fractions gave results shown in Table I. There are two very unequal zones of activity, one precipitated 0" between 35 and 45 % (v/v) acetone and a second zone of lesser activity between 50-55 %. A partial reconstruction of the whole extract was achieved by additions of equal quantities of the 45-5o%, 50-55%, 55-60% and 60-70% fractions to the 0-45% fraction. The results of t such combinations (Table II) confirm that the 50-55% fraction (almost without activity) cono0 siderably enhances the activity of the 45 % fraction when added to it. These findings are readily o 6c reproducible and have furthermore been obtained with enzyme extracts from pigs heart muscle 2 (HAPPOLD, HULLIN, NOBLE, AND SPENCER28). The progress curves for carbon dioxide production by the whole extract indicate the presence of two carbon dioxide producing steps in the reaction. The usual course of the reaction is an ~ qr...~, q - - o - - - ~ - initial rapid linear production of carbon dioxide 20 40 60 80 100 for about 3 ° minutes followed by a sharp break T/me in min~ and a subsequent linear period in which the Fig. 3- Progress curves for carbon" dioxide production by the acetone precipitated carbon dioxide production rate is markedly de fractions of the acetylmethylcarbinol creased. The progress curves for carbon dioxide enzyme system of A. aerogenes. -O--©--©0.2 ml o-450/o fraction, production by the 45% and 5o-55% fractions --(~, | ) - - ~ - - 0 . 2 ml 5o-55% fraction alone and when in combination (Fig. 3) indicate and - e - - e - - o o.2 ml 45% fraction that some separation of these two hypothetical plus 0.2 ml 55% fraction steps is occurring. The stimulating effect of the 55 % fraction on the carbon dioxide production of the 45 % fraction is limited to increasing the initial rate of carbon dioxide production only.
jO'
160
jO"
Sy
A?
Re[erences p. 556.
VOL. 8 (1952)
ENZYMICFORMATION OF ACETYLMETHYLCARBINOL
549
TABLE II CARBON
DIOXIDE
COMBINATIONS
AND
ACI~TYLMNTHYLCARBINOL
OF THE
ACETONE
ACTIVITIES
PRECIPITATBD
OF
FRACTIONS
Composition of Reaction Mixtures: o.5 ml (io mg/ml) sodium pyruvate 1.8 ml M / I 5 phosphate buffer, pH 5.8 0.2 ml 45% #action with additions o[ 0.2 ml o] #action as stated
0-45 % 0-45% 0-45% 0-450/o o-45% 0-45%
Carbon dioxide (ttM/6o min/o.2 ml)
Acetylmethylcarbinol (ttM/6o min/o.2 ml)
4.5 4.9 6.9 7.2 6.4 6.5
2.9 3.2 3.4 3.6 3.2 3.2
alone + 45-50% + 5°-55% + 55-60% + 6o-65% + 65-70%
Fresh M/I 5 phosphate buffer (pH 5.9) extract of acetone dried A. aerogenes (volume io ml) Added to
acetone 45%
PRECIPITATE Redissolved in Io ml buffer. 2.0 ml retained (Ist FRACTION
SUPERNATANT (discarded)
To remainder Added to
acetone 45%
PRECIPITATE Redissolved in 8 ml buffer. 2.0 ml retained (2nd FRACTION)
SUPERNATANT (discarded)
To remainder Added to
acetone 45%
PRECIPITATE SUPERNATANT Redissolved in 6 ml buffer. (discarded) 2.o ml used for activity test (3rd FRACTION) Fig. 4- Scheme for fractionation and further separation of the 45% acetone precipitated fraction of the acetylmethylcarbinol enzyme system of A. aerogenes. T h e 45% fraction was f u r t h e r s e p a r a t e d b y s u b m i t t i n g it to t h e f r a c t i o n a t i o n scheme shown in Fig. 4. T h e a c t i v i t y figures for the fractions t o g e t h e r w i t h t h e r e l a t i v e a m o u n t s of p y r u v a t e u t i l i z e d (Table III) were o b t a i n e d a t the end of the reactions as i n d i c a t e d b y a cessation of c a r b o n d i o x i d e e v o l u t i o n (14o minutes). R e p e a t e d p r e c i p i t a t i o n of t h e fraction leads to a m a r k e d decrease in p y r u v a t e utilization, a c e t y l m e t h y l c a r b i n o l a n d c a r b o n d i o x i d e f o r m a t i o n ; it was found impossible to r e p r e c i p i t a t e t h e f r a c t i o n m o r e t h a n twice a n d still r e t a i n a p p r e c i a b l e a c t i v i t y . The m o l a r r a t i o b e t w e e n the carbon dioxide and acetylmethylcarbinol produced remained constant but pyruvate u t i l i z a t i o n does n o t decrease a t t h e same rate, w i t h the result t h a t t h e m o l a r r a t i o p y r u v a t e u t i l i z e d : c a r b o n d i o x i d e p r o d u c e d , increases as follows: whole e n z y m e 1.5; References p. 556.
55 °
F.C.
HAPPOLD,
C. P . S P E N C E R
VOL. 8
(1952)
Ist fraction, 1.8, 2nd fraction, 3.2. Reprecipitation of the 45 % fraction results in the concentration of a p y r u v a t e utilizing enzyme which does not form acetylmethylcarbinol or carbon dioxide. TABLE III A C T I V I T I E S OF T H E FRACTIONS OBTAINED ON F U R T H E R S E P A R A T I O N OF T H E ACETONE P R E C I P I T A T E D FRACTION
45%
Fraction
Pyruvate utilization (l~M#4 o min/I.o ml)
Carbon dioxide production (t~M/I4Omin/I.O ml)
A cetylmethylcarbinol production (#M#4o min/i.o ml)
17.2 14.o 5.8 o.i
i 1.5 7.6 1.8 nil
5.6 3.9 I. I nil
Whole enzyme ist. fraction 2nd. fraction 3rd. fraction
Carbon balance studies with the enzyme system
The results obtained in the previous sections had frequently shown a variation in the theoretical molar ratio of 2 :I for the carbon dioxide and acetylmethylearbinol formed at various times during the course of the reaction. I n addition the fractionation studies had shown the possibility of disproportionate p y r u v a t e utilization. SILVERmAN AND WERKMAN.2 using large scale reaction mixtures had been able to demonstrate a molar ratio of 2 : 2 : 1 for p y r u v a t e utilized, carbon dioxide and acetylmethylcarbinol produced at the end of the reaction. A serial analysis of carbon dioxide and acetylmethylcarbinol production during the course of the enzymic reactions with the unfractionated extracts gave the results shown in Table IV. The theoretical ratio of 2 : 1 for carbon dioxide and acetylmethylearbinol is only reached after about three hours incubation when the reaction was in fact complete; i.e. the acetylmethylcarbinol formation lags behind t h a t of the carbon dioxide during the course of the reaction. The reverse effect is apparent with the o-45 % fraction (Fig. 2 and Table I I ) ; this fraction when isolated gives carbon dioxide acetylmethylcarbinol ratios of 1.5 :I, 1.5 :I and 1.4:1 after 6o minutes incubation, when the 5 o - 5 5 % fraction is present in addition to the 45 % fraction, the observed ratio then becomes 2.o: i and 1.9:1 and thus approximates to the theoretical. TABLE IV SERIAL ANALYSIS OF CARBON D I O X I D E AND A C E T Y L M E T H Y L C A R B I N O L P R O D U C T I O N D U R I N G T H E COURSE OF T H E ENZYMIC REACTION
Composition of Reaction Mixtures: 0.5 ml (5.0 mg/ml) sodium pyruvate 1.7 ml M/I 5 phosphate buffer, pH 5.9 0.5 ml enzyme extract Time (in minutes)
3° 60 ioo 14o i8o References p. 556.
Carbon dioxide A eetylmethylcarbinol produced produced (pM) (l~M)
3.8 5.6 6.5 6.7 6. 7
1.5 2.1 2.9 3.2 3.3
Molar ratio CO2:A MC
2.54 2.67 2.24 2.1o 2.03
VOL. 8 (1952)
ENZYMICFORMATION OF ACETYLMETHYLCARBINOL
551
Possible intermediates in the enzymic reaction T h e t h r e e c o m p o u n d s m o s t likely to occur as i n t e r m e d i a t e s seemed to be acetaldehyde, lactic a n d acetic acids. T h e w o r k of WERKMAN 22 a n d his associates showed t h a t a c e t a l d e h y d e as such was n o t i n v o l v e d in t h e reaction. L a c t i c acid can n o t be d e t e c t e d in s a m p l e s from large scale r e a c t i o n m i x t u r e s a t a n y t i m e d u r i n g t h e r e a c t i o n b y either the t h i o p h e n e r e a c t i o n of HOrKINS or UFFELMANN'S reagent. Similarly, a f t e r r e m o v i n g interfering ions b y the p r o c e d u r e of HUTCHENS AND KASS2s n e g a t i v e results are o b t a i n e d in t h e l a n t h a n u m n i t r a t e t e s t for acetate. No volatile acids can be d e t e c t e d in t h e distillate after r e m o v a l of p y r u v i c acid b y redistillation from an acid m a g n e s i u m mercuric sulphate mixture. T h e effect of b o t h a c e t a l d e h y d e acetic a n d lactic acids on 240 10) f t h e e n z y m i c r e a c t i o n was investig a t e d . T h e results confirm t h e 2O0 O f findings of SILVEI~MAN AND I-O--O ....-0/ WERKMAN t h a t acetaldehyde f does n o t e n t e r into t h e reaction. ~ L a c t a t e is w i t h o u t effect e i t h e r _~ u p o n t h e r a t e s of f o r m a t i o n or ._~ 120 final a m o u n t s of c a r b o n dioxide and acetylmethylcarbinol formed ~ or u p o n t h e a m o u n t of p y r u v a t e Q. ~ 86 utilized, in a d d i t i o n t h e e n z y m e s y s t e m is w i t h o u t effect u p o n 40 ' l a c t a t e alone. A c e t a t e , on t h e other hand, stimulates both the 0 20 40 60 80 100 q20 r a t e a n d a m o u n t of carbon diTime m rain= oxide a n d a c e t y l m e t h y l c a r b i n o l p r o d u c t i o n (Fig. 5 a n d T a b l e V), Fig. 5. The effect on carbon dioxide production of the addition of acetate to pyruvate dissimilations by the acetyli t s effect on t h e f o r m e r being methylcarbinol enzyme system of A. aerogenes. l i m i t e d to increasing t h e initial -O--O~O23/~M pyruvate, r a t e of c a r b o n d i o x i d e f o r m a t i o n . -~, , - - e - 23 p M pyruvate plus 2o/~M acetate.
.o//
TABLE V EFFECT
OF ADDITIONS
AMOUNTS
OF ACETATE
OF CARBON DIOXIDE
TO P Y R U V A T E AND
DISSIMILATIONS
ACETYLMETHYLCARBINOL
ON T H E
TOTAL
FORMED
Composition of Reaction Mixtures: 0. 5 ml (5.0 mg/ml) sodium pyruvate o. 5 ml enzyme extract 1.2 ml M/I5 phosphate buffer, pH 5.9
Addendum 0. 5 ml phosphate 0. 5 ml phosphate 0. 5 ml phosphate 0. 5 ml phosphate o. 5 ml phosphate 0. 5 ml phosphate
References P. 556.
buffer buffer buffer buffer + 0. 5 ml (20 pM/ml) acetate buffer + o. 5 ml (2o pM/ml) acetate buffer + o. 5 ml (20 pM/ml) acetate
Carbon dioxide produced (pM)
A cetylmethylcarbinol produced (aM)
7.6 7.2 7.3 8.9 9.0 9.2
3.7 3.6 3.7 4.2 4.2 4.5
552
VOL. 8
F . C . HAPPOLD, C. P. SPENCER
(I952)
No carbon dioxide or acetylmethylcarbinol are formed by the system from acetate alone. The "catalytic" effect of acetate on the enzymic reaction was further demonstrated by an examination of the effect of acetate concentration on the magnitude of the stimulation produced. The results shown in Fig. 6 are typical of those obtained. With the sample of the extract used, as little as 2/~M of acetate produces a maximum effect. Above this concentration the magnitude of the stimulation is largely independent of the acetate concentration though an additional stimulation is invariably noticed when the molar acetate concentration approaches that of the pyruvate.
I
100
o
Q.
j
L
•~ 8.O(
a ~ O
°) ~u
~
2.0
~'
o
5.0
10.0 150 200 Acetate added in ~1~
I
2riO
300
Fig. 6. The effect of concentration of acetate on carbon dioxide - O - - O - - O and acetylmethylcarbinol production - e - - e - - e by enzyme system from A. aerogenes during dissimilation of 2 3/~M of pyruvate.
DISCUSSION
Since these studies were completed JuNI 3° briefly reports a more complete fractionation of the enzyme from A. aerogenes. Of the two fractions he obtained, one acts on pyruvic acid to form a-acetolactic acid and carbon dioxide and the other decarboxylates this to yield acetylmethylcarbinol and carbon dioxide, the latter is without effect upon pyruvic acid. In ~:ddition, a variety of Vosges Proskauer positive organisms and the acetylmethylcarbinol enzyme system from pigs heart have been shown to be able to decarboxylate a-acetolactic acid (WATT AND KRAMPITZ3Z; KRAMPITZ AND EVERETTa*) whilst organisms which do not ordinarily produce acetylmethylcarbinol are capable of catalysing this reaction. The first "pyruvate decarboxylase" fraction has been shown to be cocarboxylase dependent whilst the a-acetolactic acid decarboxylase system is dependent upon manganese or magnesium ions but not cocarboxylase. This work leaves little doubt that the synthesis of acetylmethylcarbinol can occur through a-acetolactic acid as an intermediate. OCHOA3~ is of the opinion that the mechanism of acetylmethylcarbinol synthesis is best explained by the following reactions: Re/erences p. 556.
VOL. 8 (1952)
ENZYMIC FORMATION OF ACETYLMETHYLCARBINOL
a. Pyruvic acid + Enzyme
COs + Acetylmethylcarbinol ~ b. Pyruvic acid + Enzyme
,
>
553
(Acetaldehyde-enzyme) + CO2 + Pyruvic Acid Acetolactic acid + Enzyme
> (Acetaldehyde-enzyme) + COn Acetaldehyde + Enzyme + (Acetaldehyde-enzyme) Acetylmethylcarbinol + Enzyme
There is evidence against the occurrence of acetaldehyde as an intermediate in the bacterial enzymic synthesis of acetylmethylcarbinol and the formation of an enzyme complex is postulated to explain this. The second mechanism is postulated to account for the condensation of added acetaldehyde by the system from yeast and animal tissues, a-acetolactic acid not occurring as an intermediate in this scheme. The results reported in this paper confirm much of the findings reviewed above. We have shown that the whole enzyme system has a resultant sharp optimum pH at about 5.9 for both acetylmethylcarbinol and carbon dioxide formation. This is in agreement with the original results of SILVERMAN AND WERKMAN22 who stated that the pH optimum for the system was in the region of 5.6 to 6.0. The results obtained by these workers were irregular and some of their preparations were apparently as active at pH 4.5 as at 5.6. Our preparations were all comparatively inactive at a pH of 4.5, considerable protein precipitation occurring below this pH. Use of half saturated potassium chloride as an extracting agent for the system and the kieselguhr effect both indicated the presence of two enzymic fractions. Similarly the relative effects of coenzyme factors and metallic ions on acetylmethylcarbinol and carbon dioxide production are in accord with the presence of two distinct systems, one cocarboxylase dependent and one manganese dependent. The partial fractionation achieved indicated the presence of a pyruvate utilizing fraction and of a second fraction that was inactive towards pyruvate in the absence of tile first fraction. It seems most probable that our second fraction is identical with the a-acetolactic decarboxylase of J u s I 8°. The kinetics of carbon dioxide production by the whole system are in agreement with the conception of two distinct decarboxylating fractions, the first being associated with the initial attack on pyruvate and the second with the a-acetolactic decarboxylase. Furthermore it appears that the decarboxylating activity of the second fraction is conditional upon prior decarboxylation of the pyruvate by a system contained in the first fraction. In contrast, however, further attempted purification of our first fraction indicates that a "pyruvate utilizing non-carbon dioxide forming" system may be present which suggests that JuNI'S first fraction may not be a single enzyme. Serial analysis of the products during the course of the reaction indicates that the theoretical molar ratio of 2 : I for carbon dioxide and acetylmethylcarhinol produced is only obtained at the end of the reaction. In addition it has been shown that acetate (but not acetaldehyde) is condensed by the bacterial enzyme system during the concurrent decarboxylation of pyruvate; and causes an increase in both the rate and amount of formation of carbon dioxide and acetylmethylcarbinol. These results are not entirely compatible with the reaction paths suggested by OCI~OA33. Hydrogen acceptors have long been known to act on the enzymic formation of Re~erences p. 556.
554
F.C. HAPPOLD, C. P. SPENCER
VOL. 8
(1952)
acetylmethylcarbinol by increasing (or in the case of yeast, initiating) its formation, and it has been supposed that they exerted an "acetaldehyde sparing" action thus providing more of the two-carbon compound for condensation to acetylmethylcarbinol. Studies with washed cell suspensions of A. aerogenes (HAPPOLD AND SPENCER26) have indicated that an oxidative reaction may be involved in the conversion of pyruvic acid to acetylmethylcarbinol and that such an oxidative reaction m a y be linked with a reductive reaction (other than the reduction of the carbinol to the glycol) which also occurs in the reaction chain. An added hydrogen donor or a hydrogen donating reaction of the glycolytic cycle is able to act in place of the normal hydrogen donating system. MARTIUS~4 has proposed a hypothesis to explain pyruvate metabolism in which he postulates that the first reaction of pyruvate in the presence of co-carboxylase is dehydrogenation, and some evidence has been presented by SOUMALINER AND JXNNES~5 that dehydrogenation is the primary phase of pyruvate breakdown prior to acetylmethylcarbinol formation by yeast. I t therefore seems possible that an oxidative decarboxylation of pyruvate occurs and that acetate (or a compound closely related to it other than acetaldehyde) is the two-carbon compound involved in acetylmethylcarbinol formation. In this case, the absence of an acetaldehyde effect with the bacterial enzyme extract m a y be due to the absence of an aldehyde oxidase. In contrast, with extracts from mammalian tissue acetaldehyde m a y have a dual role (i.e. as a source of supply of the two-carbon compound and as a hydrogen donor). This would account for the fact that although the acetate effect can be observed with mammalian enzyme extracts, the analogous acetaldehyde effect is markedly greater. If acetate is involved two possibilities arise, a-acetolactic acid could arise by interaction of acetate and lactic acid. Such a scheme would involve an oxidative decarboxylation and a reduction of pyruvate and might account for the observed effects of hydrogen donors and acceptors on the system. The fact that lactate is not condensed by the bacterial system m a y be due to its not appearing as such in the reaction (enzyme complex?). Alternatively, a condensation between acetate and acetaldehyde (as an enzyme complex) is possible: CHs I CO I OH H q CO CH a
CH 8
CH a
I
--H20
CO I * CO CH a
I
+
2H
CH.0H ~ CO CH a
-
A reaction of this nature might pass through diacetyl as an intermediate which would provide the symmetrical intermediate that the isotopic studies indicate. There is no direct evidence for the occurrence of diacetyl in this role but SOUMALINER AND JXNNES3s claim to have detected diacetyl in yeast dissimilations of pyruvic acid under conditions that make its formation by fortuitous oxidation of the carbinol unlikely. In addition, an enzyme system has been isolated from mammalian tissue which catalyses the reduction of diacetyl to acetylmethylcarbinol (STuMPF, GREEN AND ZARUDNAYA36~. The isolated system uses a second molecule of diacetyl as a hydrogen donor but it is possible that, if this system is involved in the enzymic synthesis of acetylmethylcarbinol, it might be linked to the oxidative decarboxylation of pyruvate. Previous work with
Relerences p. 556.
VOL. $
(1952)
ENZYMICFORMATION OF ACETYLMETHYLCARBINOL
555
aldehydes (NEUBERG AND HIRSCH1; BERG AND WESTERFIELD 37) m a k e s i t appear probable that the condensation of acetaldehyde involves the coupling of the aldehyde (or its derivative) with a two-carbon compound and not with pyruvate or lactate, in which case a-acetolactic acid may not be involved in this pathway. The bulk of the evidence at the present time seems to indicate that this is the case, the kinetic data for carbon dioxide production on additions of acetate indicating that the increased rate involves the decarboxylation associated with the first fraction and not the aacetolactic decarboxylase fraction. To explain the increased rate of carbon dioxide formation on addition of acetate it is necessary to assume that the condensation between pyruvate and the acetaldehyde-enzyme complex is the rate limiting step, added acetate thus being able to compete with the excess pyruvate. OCHOA'S scheme for the intervention of acetaldehyde (reaction scheme b) may be redrafted as below: substituted
Pyruvic acid + Enzyme Pyruvic acid
) (Acetaldehyde-enzyme) + CO S
+ 1~ O3 ) Acetate + CO s
(Acetaldehyde-enzyme) + Acetate
Acetaldehyde
[
> Diacetyl + Enzyme
I 2H Acetylmethylcaxbinol
Such a modification provides an explanation of the effects of acetate and of hydrogen donors on the reaction. It does not appear that this mechanism operates through a-acetolactic acid as an intermediate and hence it must be supposed to function as an alternative and in addition to that reaction path. The authors wish to thank the Medical Research Council for a grant made in support of this work. SUMMARY A cell-free enzyme extract which catalyses the anaerobic decaxboxylation of pyruvic acid with the formation of acetylmethylcarbinol has been isolated from Aerobacter aerogenes b y three methods (viz. b y grinding with powdered glass, b y shaking with glass beads and b y extraction of a n acetone dried preparation). Adsorption of the complex, fractionation studies and an examination of the effects of coenzyme factors indicate t h a t a t least two systems are involved. The whole system is able to condense acetate b u t not acetaldehyde during the concurrent decarboxylation of pyruvate. I t is suggested t h a t acetate m a y be involved in one type of enzymic synthesis of acetylmethylcarbinol and t h a t acetaldehyde is condensed only after oxidation to acetate. The possibility of the occurrence of diacetyl as an intermediate in the reaction involving acetate is discussed. R~SUM]~ Un suc enzymatique d6pourvu de cellules agissant comme catalisateur de la d6carboxylation ana6robique de l'acide pyruvique avec la production d'acdtylmethylcarbinol a 6t6 obtenu de l'Aerobaaer aerogenes au moyen de trois m~thodes: en b r o y a n t les bact6ries avec du verre pulveris~, en les agitant avec des billes de verre, ou enfin par l'extraction d'une preparation sech~e avec de l'acdtone. Les donn~es fonrnies: par l'adsorption de ce suc enzymique, par les r~sultats du fractionnement et l'effet des agents activateurs indiquent qu'au moins deux syst~mes enzymatiques y sont present. L'ensemble est capable de condenser l'acdtate mais non pas l'ac6taldehyde lors de la d~carboxylation simultan~e du pyruvate. On peur supposer que l'ac~tate intervient dans un genre de synth~se enzymatique d'acdtylmethylcarbinol et que l'acdtaldehyde est condens6 seulement apr~s avoir 6t6 transform6 en ac$tate par oxydation. La possibilit6 de la presence du diacetyl comme interm6diaire dans cette rdaction est discut~e.
Re/erences p. 556.
556
F . C . HAPPOLD, C; P. SPENCER
VOL. 8
(1952)
ZUSAMMENFASSUNG E i n zellenfreier E n z y m s a f t , welcher die a n a e r o b e D e c a r b o x y l a t i o n der B r e n z t r a u b e n s A u r e u n t e r B i l d u n g v o n Acetoin katalisiert, w u r d e a u s d e m Aerobacter aerogenes a u f d r e i f a c h e m Wege, n~kmlich d u t c h ZerTeiben m i t p u l v e r i s i e r t e m Glass, d u r c h Schfitteln m i t Glassperlen u n d d u t c h E x t r a h i e r e n eines m i t A c e t o n g e t r o c k n e t e n P r ~ p a r a t e s e r h a l t e n . Die A d s o r p t i o n des E n z y m s a f f e s , die E r g e b n i s s e der F r a k t i o n i e r u n g , sowie die B e o b a c h t u n g e n iiber die W i r k u n g der A k t i v a t o r e n weisen d a r a u f hin, d a s s w e n i g s t e n s zwei E n z y m s y s t e m e dabei beteiligt sind. D a s S y s t e m als G a n z e s ist i m s t a n d e A c e t a t , n i c h t a b e r A c e t a l d e h y d wXhrend der gleichzeitigen D e c a r b o x y l a t i o n der B r e n z t r a u b e n s ~ u r e zu k o n d e n s i e r e n . Es wird a n g e n o m m e n , d a s s d a s A c e t a t in einer der e n z y m a t i s c h e n S y n t h e s e n des Acetoins beteiligt sein k 6 n n t e u n d d a s s A c e t a l d e h y d erst n a c h O x y d a t i o n zu A c e t a t k o n d e n s i e r t wird. Die M6glichkeit des A u f f r e t e n s v o n D i a c e t y l als einer Z w i s c h e n s t u f e in dieser R e a k t i o n wird erOrtert. REFERENCES 1 c. NEUBERG AND J. HIRSCH, Biochem. Z., 115 (1921) 282. 2 C. N~UBERG AND L. LEIB]~RMANN, Biochem. Z., 121 (1921) 311, 3 C. NEUBERG AND H. OHLE, Biochem. Z., 127 (1922) 327 . C. NEUBERG AND H. OHLE, Biochem. Z., 128 (1922) 61o. s C. NEUBERG AND E. REINFURTH, Biochem. Z., 143 (1923) 553. e C. NEUBERG AND A. MAY, Biochem. Z., 14o (1923) 299. 7 C. NEUBERG AND O. ROSENTHAL, Ber., 5 7 B (1924) 1436. s A. J. KLUYVER AND H. J. L. DONKER, Proc. Acad. Sci. Amsterdam, 28 (1924) 314 . 9 y . TOMIYASU, Biochem. Z., 289 (I936) 9710 y . TOMIYASU, Rept. Japan Assoc. Advancement Sci., 16 (1942) 552. 11 A. KuzlN, Chemical Abstracts, 31 (1937) 5386/9. 11 W. DIRSCHERL, Z. physiol. Chem., 188 (193o) 225. 13 W. DIRSCHERL, Z. physiol. Chem., 252 (1938) 7 o. 14 B. TANKO AND L. MUNK, Z. physiol. Chem., 262 (1939) 144. 15 N. H. GROSS AND C. H. WERKMAN, Antonie van Leeuwenhoeck. J. Microbiol. Serol., 12 (1947) 19. 16 N. H. GROSS AND C. H. WERKMAN, Arch. Biochem., 15 (1947) 125. 17 D. E. GREEN, W . W. W~STERFIELD, B. VENNESLAND, AND W . E. KNOX, J . Biol. Chem., 145 (1942 ) 69. is M. M. BARRITT, J . Path. Bact., 44 (1937) 679. 19 M. N. MICHELSON AND C. H. WERKMAN,J, Bact. 37 (1939) 619., 20 H. REYNOLDS, B. J. JACOBSSON, AND C. H. WERKMAN, J. Bact., 34 (1937) 15. 21 H. G. STAHYL AND C. H. WERKMAN, Biochem. J., 36 (1942) 575. $2 N. SILVERMAN AND C. H. WERKMAN, J. Biol. Chem., 138 (1941) 35. 23 H. TAUBER, J. Biol. Chem., 125 (1938) 191. F. C. HAPPOLD AND C. P. SPENCER, Biochim. Biophys. Acta, in t h e press. 35 T. E. FRIEDMANN AND G. E. HAUGEN, J. Biol. Chem., 147 (1943) 415 • R. MARKHAM, Biochem. J., 36 (1942) 790. 27 E. F, GA.LE, The Chemical Activities o/Bacteria, (1947). U n i v e r s i t y T u t o r i a l Press, L o n d o n . 3s F. C. HAPPOLD, R. P. HULLIN, R. L, NOBLE, AND C. P. SPENCER, (unpublished). 29 j . O. FIUTCHENS AND B. M. KASS, J . Biol. Chem., 177 (1949) 571. 30 E. JUNI, Federation Proc., 9 (195 o) 396. 31 D. WATT AND L. O. KRAMPITZ, Federation Proc., 6 (1947) 3 ol. 33 L. O. KRAMPITZ AND J. EVERETT, Arch. Biochem., 17 (1948) 8I. 33 S. OCHOA, Physiol. Rev., 31 (1951) 56. 34 C. MARTIUS, Z. physiol. Chem., 279 (1943) 96. 35 H. SUOMALAINEN AND L. JANNES, Nature, 157 (1946) 336. as p. K. STUMPF, D. E. GREEN, AND K. ZARUDNAYA, J. Biol. Chem., I67 (1947) 811. 37 R. L. BERG AND W . W . WESTERFIELD, J. Biol. Chem., 152 (1944) xi3Received,
June
i6th,
1951